Project summary Differences in gene expression are major contributors to diversity across individuals and between species. These differences have a strong genetic basis, and genetic variants that modulate gene expression, termed expression quantitative trait loci (eQTL), affect susceptibility to human diseases. Studies have found that most eQTL are tissue specific. Thus, studying tissue-specific gene regulation is critical for understanding the genetics of many human diseases. When comparing different species, changes in tissue specificity of gene expression have been found to contribute to adaptive evolution. Despite its central importance for both disease and basic genetic research, major challenges remain for studying tissue-specific gene expression. These include the high cost associated with genomic study in multiple tissues, and the association of many diseases with rare cell types that are not readily separable from the rest of the tissue. single-cell RNA sequencing (scRNA-seq) offers to significantly mitigate both of the above issues, by allowing identification of tissue and cell-type identity directly from scRNA-seq measurements. Dr. Ben David will establish the nematode worm C. elegans as a platform for studying tissue-specific gene expression within and between species. During the K99 phase of the research (Aim 1), Dr. Ben David will perform scRNA-seq based eQTL mapping across tissues of C. elegans. The study will establish experimental, statistical and computational methods for scRNA-seq based tissue-specific eQTL mapping, and the results will guide future studies in other organisms including humans. During the R00 phase of the proposal (aim 2), Dr. Ben David will use the training gained in the K99 phase and move beyond C. elegans to comparatively study other nematode species using scRNA-seq, taking advantage of the highly conserved anatomy between them. Combining scRNA-seq with the extensive experimental toolkit in nematodes, Dr. Ben David will establish C. elegans and related species as a comparative platform for investigating how gene regulation is encoded in the genome, how it changes in evolution, and the phenotypic consequences of these changes. During the K99 phase, Dr. Ben David will be mentored by Dr. Leonid Kruglyak (eQTL mapping and quantitative genetics), and advised by Dr. Aldons J. Lusis (eQTL mapping), Dr. Xia Yang (scRNA-seq experimental and analytical approaches) and Dr. Elissa Hallem (nematode neurobiology and behavior). Together with an extensive training program that include intra and extramural instruction, this diverse group of distinguished experts will provide the comprehensive training Dr. Ben David will use to accomplish the aims of the proposal, as well as to establish an independent research career.